Identification of dependable methods that allow for the isolation and proliferation of adult stem cell populations has long been a significant obstacle in the development of cell-based therapies for human diseases. One particularly promising isolation technique is based on efflux of the DMA minor groove-binding fluorophore Hoechst 33342. Using fluorescence-activated cell sorting (FACS[unreadable]1/2), a sub-population of putative adult stem cells were originally identified in the bone marrow of mice. This rare cell fraction was termed the 'side population'or 'SP'due to a distinctive FACS[unreadable]1/2 profile that resulted from weak staining by Hoechst 33342. SP cells appeared dull compared to the majority of viable cells (called the main population or MP) due to fluorescent dye effusion through the ABCG2/Bcrp1 transporter. The expression and function of this transporter allows clearance of xenobiotic substances and is believed to be important in the maintenance of primitive cells as it is sharply down-regulated following lineage commitment. Though SP cells have been isolated from a large number of tissues in various mammalian species and appear to represent a multipotent adult stem cell population capable of differentiating toward myogenic, cardiogenic, and other cell lineages;there is currently no method to expand primary cultures of these cells. Therefore, our overall goal is to develop reproducible techniques to expand and differentiate adult bone marrow and skeletal muscle-derived side population (SP) stem cells to determine their regenerative and reparative potential for the treatment of cardiac and skeletal muscle disorders. To achieve this goal, we propose to address the following specific aims and hypotheses: 1) develop a reliable method for the preservation and expansion of side population cells (i.e. we will design a three-dimensional bioreactor system to propagate SP cells derived from two clinically-applicable adult tissue sources;namely, skeletal muscle and bone marrow), 2) determine the engraftment and reconstitution potential of the expanded SP cultures (i.e. SP expansion cultures will engraft to normal or damaged cardiac and skeletal muscle and reconstitute the entire hematopoietic system in lethally-irradiated mice), and 3) determine the conditions necessary to direct differentiation of expanded SP cells (i.e. we will devise approaches for the cardiogenic and myogenic specification of skeletal muscle and bone marrow-derived SP cells that have been expanded in culture.